Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Autor(es)

Feitosa, W. B. ^[1] ; Mendes, C. M. ^[1] ; Milazzotto, M. P. ^[1] ; Rocha, A. M. ^[1] ; Martins, L. F. ^[1] ; Simoes, R. ^[1] ; Paula-Lopes, F. F. ^[1] ; Visintin, J. A. ^[1] ; Assumpcao, M. E. O. A. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Coll Vet Med & Anim Sci, Dept Anim Reprod, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Theriogenology; v. 74, n. 4, p. 563-568, SEP 1 2010.
• Citações Web of Science: 12

Resumo

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 X 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. (C) 2010 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 03/10234-7 - Espermatozoide bovino como vetor de transgene: efeito da estrutura e do tamanho do dna exogeno na internalizacao no nucleo do espermatozoide.
• Beneficiário: Weber Beringui Feitosa
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 03/07456-8 - Estudo da integracao do dna exogeno em espermatozoides de bovinos.
• Beneficiário: Mayra Elena Ortiz D' Avila Assumpcao
• Modalidade de apoio: Auxílio à Pesquisa - Regular